Blood coagulation is a process consisting of a complex interaction of various blood components (or factors) that eventually results in a fibrin clot. Generally, the blood components participating in what is referred to as the “coagulation cascade” are proenzymes or zymogens, i.e. enzymatically inactive proteins that are converted into an active form by the action of an activator. One of these coagulation factors is factor VII (FVII).
FVII is a vitamin K-dependent plasma protein synthesized in the liver and secreted into the blood as a single-chain glycoprotein with a molecular weight of 53 kDa (Broze & Majerus, J. Biol. Chem. 1980; 255:1242-1247). The FVII zymogen is converted into an activated form (FVIIa) by proteolytic cleavage at a single site, R152-I153, resulting in two chains linked by a single disulfide bridge. FVIIa in complex with tissue factor (TF), the FVIIa complex, is able to convert both FIX and FX into their activated forms, followed by reactions leading to rapid thrombin production and fibrin formation (Østerud & Rapaport, Proc Natl Acad Sci USA 1977; 74:5260-5264).
FVII undergoes post-translational modifications, including vitamin K-dependent carboxylation resulting in ten γ-carboxyglutamic acid residues in the N-terminal region of the molecule. Thus, residues number 6, 7, 14, 16, 19, 20, 25, 26, 29 and 35 shown in SEQ ID NO:2 are γ-carboxyglutamic acids residues in the Gla domain important for FVII activity. Other post-translational modifications include sugar moiety attachment at two naturally occurring N-glycosylation sites at positions 145 and 322, respectively, and at two naturally occurring O-glycosylation sites at positions 52 and 60, respectively.
The gene coding for human FVII (hFVII) has been mapped to chromosome 13 at q34-qter 9 (de Grouchy et al., Hum Genet 1984; 66:230-233). It contains nine exons and spans 12.8 Kb (O'Hara et al., Proc Natl Acad Sci USA 1987; 84:5158-5162). The gene organisation and protein structure of FVII are similar to those of other vitamin K-dependent procoagulant proteins, with exons 1a and 1b encoding for signal sequence; exon 2 the propeptide and Gla domain; exon 3 a short hydrophobic region; exons 4 and 5 the epidermal growth factor-like domains; and exon 6 through 8 the serine protease catalytic domain (Yoshitake et al., Biochemistry 1985; 24: 3736-3750).
Reports exist on experimental three-dimensional structures of hFVIIa (Pike et al., Proc Natl Acad Sci USA 1999; 96:8925-30 and Kemball-Cook et al., J. Struct. Biol. 1999; 127:213-223), of hFVIIa in complex with soluble tissue factor using X-ray crystallographic methods (Banner et al., Nature, 1996; 380:41 and Zhang et al., J. Mol. Biol., 1999; 285: 2089), and of smaller fragments of hFVII (Muranyi et al., Biochemistry, 1998; 37:10605 and Kao et al., Biochemistry, 1999; 38:7097).
Relatively few protein-engineered variants of FVII have been reported (Dickinson & Ruf, J Biol Chem, 1997; 272:19875-19879, Kemball-Cook et al., J Biol Chem, 1998; 273:8516-8521, Bharadwaj et al., J Biol Chem, 1996; 271:30685-30691, Ruf et al., Biochemistry, 1999; 38:1957-1966).
Reports exist on expression of FVII in BHK or other mammalian cells (WO 92/15686, WO 91/11514 and WO 88/10295) and co-expression of FVII and kex2 endoprotease in eukaryotic cells (WO 00/28065).
Commercial preparations of recombinant human FVIIa (rhFVIIa) are sold under the tradename NOVOSEVEN®. NOVOSEVEN® is indicated for the treatment of bleeding episodes in hemophilia A or B patients. NOVOSEVEN® is the only rhFVIIa for effective and reliable treatment of bleeding episodes available on the market.
An inactive form of FVII in which arginine 152 and/or isoleucine 153 are modified has been reported in WO 91/11514. These amino acids are located at the activation site. WO 96/12800 describes inactivation of FVIIa by a serine proteinase inhibitor; inactivation by carbamylation of FVIIa at the α-amino acid group I153 has been described by Petersen et al., Eur J Biochem, 1999; 261:124-129. The inactivated form is capable of competing with hFVII or hFVIIa for binding to TF and inhibiting clotting activity. The inactivated form of FVIIa is suggested to be used for treatment of patients suffering from hypercoagulable states, such as patients with sepsis, at risk of myocardial infarction or of thrombotic stroke.
WO 98/32466 suggests that FVII, among many other proteins, may be PEGylated (i.e. attached to one or more polyethylene glycol molecules) but does not contain any further information in this respect.
WO 01/58935 discloses a new strategy for developing FVII or FVIIa molecules having inter alia an increased half-life by means of directed glycosylation or PEGylation.
WO 03/093465 discloses FVII or FVIIa variants having certain modifications in the Gla domain and having one or more N-glycosylation sites introduced outside the Gla domain.
A circulating rhFVIIa half-life of 2.3 hours was reported in “Summary Basis for Approval for NOVOSEVEN®”, FDA reference number 96-0597. Relatively high doses of frequent administration are necessary to reach and sustain the desired therapeutic or prophylactic effect. As a consequence, adequate dose regulation is difficult to obtain and the need for frequent intravenous administrations imposes restrictions on the patient's way of living.
In normal hemostasis, the procoagulant system is in balance with anticoagulant systems involved in the termination of the hemostatic reaction and the fibrinolytic system, which dissolves clots once they are formed. The anticoagulant systems contain several protease inhibitors, e.g., the Tissue Factor Pathway Inhibitor (TFPI), antithombin-III (AT-III), heparin cofactor-II (HC-II), and the protein C pathway.
TFPI is a reversible, active site-directed inhibitor of FXa, which regulates coagulation by inhibiting FVIIa-TF in a FXa-dependent manner. The TFPI-FXa complex binds to the FVIIa-TF complex, resulting in the formation of a TF-FVIIa-TFPI-FXa complex.
The in vivo relevance of TFPI is supported by experiments showing a hemostatic effect of a neutralizing anti-TFPI antibody in a hemophilia bleeding model (Erhardtsen et al. Blood Coagul Fibrinolysis 1995; 6:388-394). Furthermore, in biochemical reconstitution experiments, TFPI was shown to extend the initiation phase and reduce the rate of thrombin generation during the propagation phase (van't Veer and Mann; J. Biol. Chem. 1997; 272: 4367-4377).
An object of the present invention is to provide FVII or FVIIa variants which exhibit an increased clotting activity as compared to hFVIIa or rhFVIIa. It is contemplated that this may be obtained by way of FVII or FVIIa variants having an altered affinity to TFPI.
Another problem in current rhFVIIa treatment is the relative instability of the molecule with respect to proteolytic degradation. Proteolytic degradation is a major obstacle for obtaining a preparation in solution as opposed to a lyophilized product. The advantage of obtaining a stable soluble preparation lies in easier handling for the patient, and, in the case of emergencies, quicker action, which potentially can become life saving. Attempts to prevent proteolytic degradation by site directed mutagenesis at major proteolytic sites have been disclosed in WO 88/10295.
Thus, a further object of the present invention is to provide FVII/FVIIa variants which, in addition to the above-mentioned improved properties, are more stable towards proteolytic degradation, i.e. possess reduced sensitivity to proteolytic degradation.
A molecule with a longer circulation half-life would decrease the number of necessary administrations. Given the association of current FVIIa product with frequent injections, and the potential for obtaining more optimal therapeutic FVIIa levels with concomitant enhanced therapeutic effect, there is a clear need for improved FVII- or FVIIa-like molecules. One way to increase the circulation half-life of a protein is to ensure that renal clearance of the protein is reduced. This may be achieved by conjugating the protein to a chemical moiety which is capable of conferring reduced renal clearance to the protein. Furthermore, attachment of a chemical moiety to the protein or substitution of amino acids exposed to proteolysis may effectively block a proteolytic enzyme from contact leading to proteolytic degradation of the protein. Polyethylene glycol (PEG) is one such chemical moiety that has been used in the preparation of therapeutic protein products.
Thus, a further objective of the present invention is to provide FVII/FVIIa variants which, in addition to the above-mentioned improved properties, possess an increased functional in vivo half-life and/or an increased serum half-life.
The improved FVII/FVIIa variants disclosed herein address these objectives.